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United States Patent |
6,126,839
|
Kreader
,   et al.
|
October 3, 2000
|
Use of an alcohol-salt buffer wash for the efficient recovery of
mycobacteria and mycobacterial DNA from respiratory sediment
Abstract
The present invention relates to methods for concentrating bacteria from a
viscous biological sample. The methods involve adding to the sample a
water-soluble, density-lowering agent having a density of 0.7 to 0.9 g/ml
and a boiling point greater than 50.degree. C. The invention also relates
to methods for concentrating bacteria and free bacterial nucleic acids
from a biological sample that involve mixing with the sample a
density-lowering agent and a monovalent salt.
Inventors:
|
Kreader; Carol (114 Fairview Cir., Webster, NY 14580);
Backus; John W. (12865 Via Caballo Rojo, San Diego, CA 92129);
Kerschner; Joanne H. (9033 Clayton Rd., St. Louis, MO 63117);
Mehta; Rashmi (122 Machado Cove, Dona Paula, Goa, IN)
|
Appl. No.:
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013987 |
Filed:
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January 27, 1998 |
Current U.S. Class: |
210/724; 210/723; 210/725; 210/728; 210/729; 210/787; 210/789; 435/6; 435/243; 435/253.1 |
Intern'l Class: |
B01D 021/26; B01D 021/01; C12N 001/00 |
Field of Search: |
210/634,723,724,725,728,729,781,782,789,787
435/2,6,243,253.1,260,261
436/177,178
|
References Cited
Foreign Patent Documents |
0 285 439 A2 | May., 1988 | EP.
| |
0 626 456 A1 | Nov., 1994 | EP.
| |
94917192 | Jan., 1998 | EP.
| |
Other References
Beavis, K.G. et al.; Evaluation of Amplicor PCR for Direct Detection of
Mycobacterium tuberculosis from Sputum Specimens; Journal of Clinical
Microbiology, Oct., 1995, p. 2582-2586.
Williams, D.L. et al.; Ethanol Fixation of Sputum Sediments for DNS-Based
Detection of Mycobacterium tuberculosis; Journal of Clinical Microbiology,
Jun. 1995, p. 1558-1561.
Best, M. et al.; Efficacies of Selected Disinfectants against Mycobacterium
tuberculosis; Journal of Clinical Microbiology, Oct., 1990, p. 2234-2239.
Lind, A., et al.; A carrier method for the assessment of the effectiveness
of disinfectants against Mycobacterium tuberculosis; Journal of Hospital
Infection (1986) 7, 60-67.
Kent, P.T., et al.; Public Health Mycobacteriology A Guide for the Level
III Laboratory; U.S. Dept. of Health and Human Services Public Health
Service Centers for Disease Control, 1985, p. 31-47.
Noordhoek, G.T., et al.; Sensitivity and Specificity of PCR for Detection
of Mycobacterium tuberculosis: a Blind Comparison Study among Seven
Laboratories; Journal of Clinical Microbiology, Feb. 1994, p. 277-284.
Nolte, F.S. et al.; Direct Detection of Mycobacterium tuberculosis in
Sputum by Polymerase Chain Reaction and DNA Hybridization; Journal of
Clinical Microbiology, Jul. 1993, p. 1777-1782.
Forbes, B.A. et al.; Direct Detection of Mycobacterium tuberculosis in
Respiratory Specimens in a Clinical Laboratory by Polymerase Chain
Reaction; Journal of Clinical Microbiology, Jul. 1993, p. 1688-1694.
Hurley, S.S. et al.; Rapid Lysis Technique for Mycobacterial Species;
Journal of Clinical Microbiology, Nov. 1987, p. 2227-2229.
Buck, G.E., et al.; Rapid, Simple Method for Treating Clinical Specimens
Containing Mycobacterium tuberculosis To Remove DNA for Polymerase Chain
Reaction; J. of Clin. Microbiology, May 1992, p. 1331-1334.
Reischl,U. et al.; PCR-Based Detection of Mycobacteria in Sputum Samples
Using a Simple and Reliable DNA Extraction Protocol; BioTechniques, vol.
17, No. 5 (1995) , p. 844-845.
Zambardi, G. et al.; Comparison of three primer sets for the detection of
Mycobacterium tuberculosis in clinical samples by polymerase chain
reaction; Am Biol Clin (1993), 50, p. 893-897.
Folgueira, L. et al.; Detection of Mycobacterium tuberculosis DNA in
Clinical Samples by Using a Simple Lysis Method and Polymerase Chain
Reaction; Journal of Clinical Microbiology, Apr. 1993, p. 1019-1021.
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Primary Examiner: Kim; John
Parent Case Text
This application claims the benefit of U.S. Provisional Application Ser.
No. 60/039,239, filed on Feb. 28, 1997.
Claims
What is claimed:
1. A method for concentrating bacteria and free bacterial nucleic acids
from a biological sample comprising:
a) adding to the sample, in an amount sufficient to reduce the density of
the sample to less than the density of the bacteria, a water-soluble,
density-lowering agent having a density of 0.7 to 0.9 g/ml and a boiling
point greater than 50.degree. C., and in an amount sufficient to
precipitate the free bacterial nucleic acids, a monovalent salt; and
b) centrifuging the sample to pellet any bacteria and any free bacterial
nucleic acids present in the sample.
2. The method according to claim 1 wherein the sample is pretreated with
reagents that interfere with nucleic acid analysis.
3. The method according to claim 2 wherein the sample is a respiratory
sediment.
4. The method according to claim 1 wherein the biological sample is
selected from the group consisting of: respiratory sample, respiratory
sediment, oral fluid, vaginal fluid, seminal fluid, wound infection fluid,
and abcess fluid.
5. The method according to claim 1 wherein the density-lowering agent is
selected from the group consisting of: substituted or unsubstituted methyl
alcohol, substituted or unsubstituted ethyl alcohol, substituted or
unsubstituted propyl alcohol, and ketones.
6. The method according to claim 1 wherein the density-lowering agent is
selected from the group consisting of: substituted or unsubstituted methyl
alcohol, substituted or unsubstituted ethyl alcohol, and substituted or
unsubstituted propyl alcohol.
7. The method according to claim 6 wherein the density-lowering agent is
ethanol.
8. The method according to claim 1 wherein 2 to 6 volumes of the
density-lowering agent per unit volume of sample is added.
9. The method according to claim 8 wherein 2 to 2.5 volumes of the
density-lowering agent per unit volume of sample is added.
10. The method according to claim 1 wherein the bacteria is selected from
the group consisting of: Mycobacterium sp., Prevotella sp., Porphyromonas
sp., Chlamydia trachomatis sp., Neisseria gonorrhoeae sp., Staphylococcus
sp., Streptococcus sp., Enterococcus sp., Clostridium sp., and Bacteroides
sp.
11. The method according to claim 10 wherein the bacteria is selected from
the group consisting of: Mycobacterium tuberculosis complex and
Mycobacterium avium complex.
12. The method according to claim 1 wherein the monovalent salt is selected
from the group consisting of sodium acetate, sodium chloride, ammonium
acetate, and lithium chloride.
13. The method according to claim 1 wherein the bacteria is Mycobacterium
tuberculosis, the viscous biological sample is respiratory sediment, the
density-lowering agent is ethanol, and the monovalent salt is sodium
acetate.
14. A method for concentrating bacteria from a viscous biological sample
comprising:
a) adding to the sample in an amount sufficient to reduce the density of
the sample to less than the density of the bacteria a water-soluble,
density-lowering agent having a density of 0.7 to 0.9 g/ml and a boiling
point greater than 50.degree. C., and a pH buffering agent; and
b) centrifuging the sample to pellet any bacteria present in the sample.
15. The method according to claim 14 wherein the pH buffering agent brings
the pH of the sample to between 6 and 9.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for concentrating and recovering
bacteria and free bacterial nucleic acids from biological samples. In
particular, the present invention relates to methods for concentrating
Mycobacterium tuberculosis in a manner that is compatible with subsequent
nucleic acid analysis.
2. Background Information
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in
humans, is conventionally identified by time-consuming microbiological
culture. Clinical specimens submitted for mycobacterial culture are often
contaminated with other more rapidly growing microorganisms. These
specimens, typically a sputum or other respiratory sample, must be
subjected to a digestion-decontamination process to liquefy viscous
organic material and eliminate unwanted organisms. The most common
reagents used in the digestion-decontamination process are
N-acetyl-L-cysteine-sodium hydroxide (NALC--NaOH), sodium hydroxide-sodium
dodecyl sulfate (NaOH--SDS) or NaOH alone. A typical
digestion-decontamination protocol would include incubating the
respiratory sample with one of the above reagents, lowering the pH of the
mixture by diluting with buffer solution or water, and centrifuging the
mixture to concentrate the mycobacteria in a pellet. A portion of the
supernatant would be decanted and the pellet resuspended in the remaining
supernatant fluid or in a buffer solution. Suspensions obtained in such a
manner are termed "respiratory sediments".
While respiratory sediments are suitable for culture, they are not suitable
for nucleic acid analysis, such as nucleic acid amplification, restriction
digestion, and nucleotide sequencing. Prior to conducting nucleic acid
analysis, it is necessary to remove the digestion-decontamination reagents
from the respiratory sediments because these reagents interfere with
nucleic acid analysis. It is also useful to further concentrate the
mycobacteria from sediment samples having low mycobacteria titers prior to
nucleic acid analysis to increase the likelihood of nucleic acid
detection.
Prior to the present invention, those skilled in the art attempted to solve
the problems of contaminating digestion-decontamination reagents and low
bacterial concentrations by diluting the samples in aqueous solutions,
further centrifuging to pellet the mycobacteria, and discarding the
supernatant solution containing the digestion-decontamination reagents.
For example, Beavis et al. (J. Clin. Microbiol., 33, 2582-2586 (1995)) use
a method in which 100 .mu.L of respiratory sediment is mixed with 500
.mu.L of a specimen wash reagent comprising Tris-HCl and 1% solubilizer.
The mycobacteria are pelleted from the mixture by centrifugation at
12,500.times.g for 10 minutes. (See also, Roche Molecular Systems. 1994.
Roche Amplicor Mycobacterium tuberculosis test insert, Roche Molecular
Systems, Branchburg, N.J.) Such methods, however, have limitations. These
methods are not particularly suitable for concentrating bacteria from
large sample volumes because addition of the wash buffer increases the
volume five-fold. In addition, such methods are limited because
mycobacteria are very buoyant and are difficult to pellet from aqueous
media. Thus, centrifugation in water or aqueous buffer solution, such as
that utilized by Beavis et al., can result in significant loss of the
target organisms. In addition, free mycobacterial nucleic acids in the
respiratory sediment are not pelleted during centrifugation in aqueous
medium. Free nucleic acids exist in the respiratory sediment because they
can be released during bacterial lysis caused by digestion and
decontamination of fresh samples, and by freezing and thawing of stored
respiratory sediments.
The present invention overcomes these problems by providing a method for
concentrating bacteria, including M. tuberculosis, from viscous biological
samples. The present invention also provides a method for concentrating
free bacterial nucleic acids as well as the bacteria present in biological
samples. Samples processed according to the methods of the present
invention can be used for subsequent nucleic acid analysis.
SUMMARY OF THE INVENTION
Thus, it is an object of the present invention to provide a method for
concentrating and processing bacteria that removes
digestion-decontamination reagents from the sample.
It is another object of the present invention to simultaneously recover
both bacteria and any free bacterial DNA present in the biological sample.
It has been unexpectedly found that a water-miscible, density-lowering
agent having a density of 0.7 to 0.9 g/ml and a boiling point greater than
50.degree. C. can be used to concentrate bacteria from biological samples.
It has also been found that such density-lowering agents can be used in
combination with a monovalent salt, such as sodium acetate, to recover
intact bacteria and bacterial DNA from biological samples that have a
density greater than that of the target bacteria.
In one embodiment, the present invention relates to a method for
concentrating bacteria from a viscous biological sample. The method
comprises adding to the sample, in an amount sufficient to reduce the
density of the sample to less than the density of the bacteria, a
water-soluble, density-lowering agent having a density of 0.7 to 0.9 g/ml
and a boiling point greater than 50.degree. C., and centrifuging the
sample to pellet any bacteria present in the sample.
In another embodiment, the present invention relates to a method for
concentrating bacteria and bacterial nucleic acids from a biological
sample. The method comprises adding to the sample, in an amount sufficient
to reduce the density of the sample to less than the density of the
bacteria, a water-soluble, density-lowering agent having a density of 0.7
to 0.9 g/ml and a boiling point greater than 50.degree. C., and, in an
amount sufficient to precipitate the free bacterial nucleic acids, a
monovalent salt. The sample is then centrifuged to pellet any bacteria and
any free bacterial nucleic acids present in the sample.
In a further embodiment, the present invention relates to a method for
concentrating bacteria from a viscous biological sample and,
simultaneously, adjusting the pH of the sample. The method involves adding
to the sample, in an amount sufficient to reduce the density of the sample
to less than the density of the bacteria, a water-soluble,
density-lowering agent having a density of 0.7 to 0.9 g/ml and a boiling
point greater than 50.degree. C., and a pH buffering agent. The sample is
then centrifuged to pellet any bacteria present in the sample.
Various other objects and advantages of the present invention will be
apparent from the detailed description of the invention.
All publications mentioned herein are hereby incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods for processing samples for detection
and identification of bacteria, such as Mycobacterium tuberculosis, that
are compatible with nucleic acid based analysis. The present methods are
compatible with a variety of nucleic acid analysis procedures, including
nucleic acid amplification (such as PCR and ligase chain reaction),
restriction digestion, and nucleotide sequencing, because the inhibitory
substances employed for digestion-decontamination of the sample are
efficiently removed.
In one embodiment, the present invention relates to methods for
concentrating bacteria from viscous biological samples. The bacteria
contained in a biological sample are concentrated by mixing the sample
with a water-miscible, density-lowering agent and then centrifuging the
sample to pellet any bacteria present in the sample. The density-lowering
agent reduces the density of the resulting mixture to less than that of
the bacteria, and thereby, enhances recovery of the bacteria upon
centrifugation.
Density-lowering agents suitable for use in the present invention are
water-soluble, have a density of 0.7 to 0.9 g/ml, and have a boiling point
greater than 50.degree. C. Such agents include, but are not limited to,
substituted or unsubstituted methyl alcohols, substituted or unsubstituted
ethyl alcohols, substituted or unsubstituted propyl alcohols, and ketones.
For example, ethanol, methanol, methyl ethyl ketone, 2-pentanol,
3-pentanone, propanol, isobutyl alcohol, 2-propanol, tert. butyl alcohol,
or 2-propanone could be employed as the density-lowering agent in the
present invention. Preferably, the density-lowering agent is a substituted
or unsubstituted methyl alcohol, substituted or unsubstituted ethyl
alcohol, or substituted or unsubstituted propyl alcohol. More preferably,
ethanol is used as the density-lowering agent in the methods of the
present invention.
The density-lowering agent is added to the sample in an amount sufficient
to reduce the density of the sample to less than that of the target
bacteria. Such amounts are readily determinable by those skilled in the
art. Preferably, 2 to 6 volumes of density-lowering agent are added per
unit volume of sample; more preferably, 2 to 2.5 volumes of
density-lowering agent are added per unit volume of sample.
In another embodiment, the present invention relates to methods for
concentrating bacteria from a viscous biological sample and,
simultaneously, adjusting the pH of the sample to a level compatible with
subsequent analysis. For example, a buffer such as acetate, citrate, or
Tris can be combined with the density lowering agent to wash sputum
sediment samples having a pH of 12-14 to lower the pH to a level
compatible with polymer capture (pH 6.8) or PCR (pH 8.5). (For a
discussion of polymer capture, see U.S. Pat. Nos. 5,582,988, 5,434,270,
and 5,523,368.) Bacteria contained in a biological sample are concentrated
by mixing the sample with a water-soluble, density-lowering agent and a pH
buffering agent. The sample is then centrifuged to pellet any bacteria
present in the sample.
In a further embodiment, the present invention relates to methods for
concentrating bacteria and free bacterial nucleic acids from biological
samples. Bacterial nucleic acids can be released from the bacteria, for
example, during storage and freeze-thaw treatments. This free bacterial
DNA in the sample can be concentrated and recovered using the present
invention. Both bacteria and free bacterial nucleic acids contained in a
biological sample are concentrated by treating the sample with a
water-soluble, density-lowering agent and a monovalent salt. The
density-lowering agent and the monovalent salt may be added to the pellet
pre-mixed or individually. The mixture is then centrifuged to pellet
bacteria and bacterial nucleic acids that may be present in the sample.
The monovalent salt helps to precipitate free DNA from aqueous solution as
it provides cations required to counter the negative charge on DNA. The
use of a density-lowering agent, such as ethanol or isopropanol, together
with a monovalent salt such as an acetate or chloride salt of sodium,
potassium, ammonium, or lithium, results in the concentration and recovery
of bacteria and free bacterial DNA present in the sample.
Monovalent salts suitable for use in the present invention include, but are
not limited to, metal salts of acetate or chloride and non-metal salts of
acetate or chloride. Preferably, the monovalent salt is a lithium, sodium,
potassium or ammonium salt of acetate or chloride. More preferably, the
monovalent salt is sodium acetate, sodium chloride, ammonium acetate, or
lithium chloride. The monovalent salt is added in an amount sufficient to
precipitate any free bacterial nucleic acids present in the sample. Such
amounts are readily determinable by those skilled in the art.
Bacteria that can be concentrated using the methods of the present
invention include, but are not limited to, mycobacteria (such as
Mycobacterium tuberculosis and Mycobacterium avium), Prevotella sp.,
Porphyromonas sp., Chlamydia trachomatis sp., Neisseria gonorrhoeae sp.,
Staphylococcus sp., Streptococcus sp., Enterococcus sp., Clostridium sp.,
and Bacteroides sp. Other detectable species would be readily apparent to
one skilled in the art.
Bacteria can be concentrated and recovered using the methods of the present
invention from various viscous biological samples including, but not
limited to, respiratory samples (such as sputum samples), respiratory
sediment, oral fluid, vaginal fluid, seminal fluid, wound infection fluid,
and abcess fluid. Preferably, the biological sample is a respiratory
sample. More preferably, the respiratory sample is pretreated to obtain a
respiratory sediment for use in the present invention.
By way of example, M. tuberculosis can be concentrated from a respiratory
sediment according to the methods of the present invention. To prepare the
respiratory sediment, a respiratory sample, such as sputum, is first
processed by conventional procedures for liquification and
decontamination. Any of the known digestion-decontamination procedures are
suitable for this step including, but not limited to, various
modifications of the NALC--NaOH treatment (Kent & Kubica, Public Health
Mycobacteriology A Guide for the Level III Laboratory, U.S. Department of
Health and Human Services, 31-47(1985); Noordhock et al., J. Clin.
Microbiol., 32, 277-284 (1994)). These methods generally include
liquifying the sample and killing competing bacteria with a
digestion-decontamination reagent and then centrifuging the liquified
sample to pellet the bacteria. The respended pellet, the respiratory
sediment, can then be processed according to the present invention to
concentrate mycobacteria present therein. For example, M. tuberculosis can
be concentrated from the prepared respiratory sediment using the
density-lowering agent ethanol.
To concentrate and recover M. tuberculosis and any mycobacterial nucleic
acids present in the respiratory sediment, the specimen can be treated
with a density-lowering agent such as ethanol, and a monovalent salt such
as sodium acetate, according to the methods of the present invention to
remove contaminating digestion-decontamination reagents, to concentrate
low bacterial titers, and to recover free mycobacterial nucleic acids.
A density-lowering agent together with sodium or ammonium acetate can be
utilized to concentrate, wash, and change to a desired value the pH of
mycobacterial preparations from respiratory sediment samples, and render
them suitable for subsequent nucleic acid detection or analysis such as
polymerase chain reaction (PCR) analysis. For nucleic acid detection
and/or analysis the recovered mycobacteria present in the pellet are lysed
using standard lysing methods well known to those skilled in the art.
Suitable lysing methods include, but are not limited to, heating,
sonication, bead beating, freeze-thaw; and protease digestion. See, for
example, Noordhock et al., J. Clin. Microbiol., 32, 277-284 (1994); Nolte
et al., J. Clin. Microbiol., 31, 1777-1782 (1993); Forbes et al., J. Clin.
Microbiol., 31, 1688-1694 (1993); Hurley et al., J. Clin. Microbiol., 25,
2227-2229 (1987); and Folgueira et al., J. Clin.
Microbiol., 31, 1019-1021 (1993).
The nucleic acids released from the lysed mycobacteria as well as the
recovered free mycobacterial nucleic acids may be used for nucleic acid
analysis as the digestion-decontamination reagents have been removed.
The recovered nucleic acids can be used, for example, in nucleic acid
hybridization methods and nucleic acid amplification procedures, such as,
PCR, ligase chain reaction, nucleic acid sequence based amplification
(NASBA), transcription mediated amplification (TMA), strand displacement
amplification (SDA), and Q-beta replicase. Such protocols are well known
in the art and are readily available to those skilled in the art. For
example, the general principles and conditions for amplification and
detection of nucleic acids using PCR are quite well known, the details of
which are provided in numerous references including U.S. Pat. Nos.
4,683,195, 4,683,202, and 4,965,188, and by Guatelli et al, Clin.
Microbiol. Rev., 2 (2), pp. 217-226 (1989). The noted U.S. patents are
incorporated herein by reference. In view of the teaching in the art and
the specific teaching provided herein, a worker skilled in the art should
have no difficulty in practicing the present invention by combining the
bacteria concentration and recovery method of this invention with cell
lysis and polymerase chain reaction procedures.
The following Examples are provided to illustrate certain embodiments of
the present invention, and are not to be construed as limiting the
invention.
EXAMPLES
Materials and Methods
Frozen sputum sediments free of or containing M. tuberculosis (Mtb) were
obtained from several clinical microbiology laboratories.
The following reagents were prepared and used in the experiments.
(Deionized water was used in the preparation of all reagents.)
1. Sodium acetate solution (NaAc):
Anhydrous sodium acetate (12.3 g) was dissolved in approximately 40 mL
H.sub.2 O. The pH of the solution was adjusted to 5.2 with glacial acetic
acid, and the volume brought to 50 mL with water.
2. Ethanol/sodium acetate reagent (EtOH/NaAc):
One part by volume of the above sodium acetate solution was combined with
20 to 25 parts by volume ethanol. Both 200 proof (punctilious) ethanol and
denatured ethanol (containing 5% isopropanol and 5% methanol) were used
with similar success.
Method 1
NaAc and EtOH Added Separately to Sputum Sediment Sample
One part by volume of the above-described NaAc solution was pipetted into a
1.5 mL microcentrifuge tube. Ten parts by volume of sputum sediment was
added and the solution was vortexed briefly to mix. Twenty to twenty-five
parts by volume of ethanol was added, and the solution was vortexed again
briefly to mix. The microcentrifuge tube containing the sample was then
placed in a microcentrifuge and spun at 16,000.times.g for 15 minutes to
pellet bacteria and precipitate nucleic acids, and the supernatant was
discarded.
For further processing, bacterial/DNA pellets were resuspended in lysis
reagent (10 mM Tris, pH 8.0, 1% Tween 20, 1% Triton X-100, 0.1% sodium
dodecyl sulfate) and heated at 100.degree. C. for 30 min to lyse bacteria
and release DNA. DNA was recovered from these lysates by the polymer
capture technique, as described in U.S. Pat. No. 5,582,988. Briefly, a
soluble, weakly basic cationic polymer is used. DNA binds
electrostatically to the polymer via its negatively charged phosphate
groups, and forms an insoluble complex. The complex is pelleted by
centrifugation, the supernatant is discarded, and the purified DNA is
released by heating in alkaline solution.
Recovered mycobacterial DNA was amplified and detected using a Johnson &
Johnson Clinical Diagnostics, Inc. pouch containment system for PCR
nucleic acid amplification and detection as described in U.S. Pat. Nos.
5,089,233, 5,229,297 and 5,380,489. Briefly, sample plus PCR reagents were
mixed and loaded into a blister of the plastic pouch, then the pouch is
sealed. Biotin-labeled oligonucleotides, representing DNA sequences unique
to MTb, are used to prime amplification of MTb-specific DNA with thermally
stable DNA polymerase. Reaction mixtures containing sample, the DNA
polymerase, dNTP's, buffer and salt are first heated to denature DNA, then
cycled back and forth from high temperature (95.degree. C.) to denature
and lower temperature (65-75.degree. C.) to anneal primers and extend or
synthesize copies of the DNA sequence. Amplified target is detected after
reaction products are forced through a detection chamber, where they
hybridize with complementary oligos attached to beads. The hybridized
product is detected after HRP-streptavidin and subsequently an HRP-dye
substrate are forced through the detection chamber.
Method 2
NaAc/EtOH Combined Reagent Mixed with Sputum Sediment Sample
Twenty to twenty-five parts by volume of the combined EtOH/NaAc reagent was
pipetted into a microcentrifuge tube. Ten parts by volume of sputum
sediment sample was added, and the solution was vortexed briefly to mix.
The microcentrifuge tube containing the sample was then placed in a
microcentrifuge and spun at 16,000.times.g for 15 minutes to pellet
bacteria and precipitated nucleic acids, and the supernatant was
discarded.
For further processing, bacterial/DNA pellets were resuspended in lysis
reagent (10 mM Tris, pH 8.0, 1% Tween 20, 1% Triton X-100, 0.1% sodium
dodecyl sulfate) and heated at 100.degree. C. for 30 min to lyse bacteria
and release DNA. DNA was recovered from these lysates by the polymer
capture technique, as described in U.S. Pat. No. 5,582,988.
Recovered mycobacterial DNA was amplified and detected using a Johnson &
Johnson Clinical Diagnostics, Inc. pouch containment system for PCR
nucleic acid amplification and detection as described in U.S. Pat. Nos.
5,089,233, 5,229,297 and 5,380,489.
Example 1
Effects of pH, Salt, Alcohol, and Temperature on Precipitation and Recovery
of Free DNA
To identify optimum conditions for recovery of free DNA from solution over
the pH range found in sputum sediment and other respiratory sediment
samples, water was adjusted to pH 7, 10, or 14 with sodium hydroxide to
simulate the pH range of respiratory sediments, and used to dilute calf
thymus DNA (Ct-DNA), obtained from Sigma Chemical Co., to a final
concentration of 50 ng DNA/.mu.L. Either sodium chloride (NaCl) or sodium
acetate (NAAC) was added to each pH adjusted DNA sample to give final
concentrations of 0.2M NaCl or 0.3M NaAc. One half milliliter aliquots
were transferred to microcentrifuge tubes, mixed with either 1 mL of
ethanol (EtOH) or 0.5 mL of isopropanol (IPP), and either immediately
centrifuged at ambient room temperature (RT) or incubated at -20.degree.
C. for 1 hr before centrifugation. Centrifugation was at 16,000.times.g
for 15 minutes. Pelleted material was resuspended in 0.5 mL of water, and
its absorption at 260 nm, the .lambda.max for DNA, was measured with a
spectrophotometer and compared with the absorption of the unprecipitated
material. The percent recovery of DNA for the different experimental
conditions is presented below in Table 1.
TABLE 1
______________________________________
Effects of pH, Salt, Alcohol, and Incubation at -20.degree. C. on
Recovery of DNA
salt = NaCl
salt = NaAc
pH of % % std dev
DNA soln alcohol incubation recovery recovery (n = 3)
______________________________________
7 EtOH NONE 13 120 0
EtOH 1 h, -20 15 120 2
IPP NONE 12 117 1
IPP 1 h, -20 86 118 0
10 EtOH NONE 16 120 1
EtOH 1 h, -20 20 119 1
IPP NONE 22 118 0
IPP 1 h, -20 54 113 4
14 EtOH NONE 6 93 1
EtOH 1 h, -20 5 92 0
IPP NONE 2 6 3
IPP 1 h, -20 2 75 3
______________________________________
The results show that NaAc combined with either EtOH or IPP is effective in
allowing quantitative recovery of DNA from samples at pH 7 or 10, whether
centrifuged immediately or after incubating at -20.degree. C. Even at pH
14, the recovery of DNA with a NaAc and EtOH combination was nearly
quantitative, but IPP in combination with NaAc was less effective at this
pH. The combination of NaCl and alcohol did not allow efficient recovery
of Ct-DNA under any of the conditions tested. Therefore, the combination
of NaAc with EtOH and immediate centrifugation after mixing with the
sample at ambient temperature are preferred for use with sputum sediment
and other respiratory sediment samples.
Example 2
Recovery of M. Tuberculosis (Mtb) from Sputum Sediment
In this experiment, the recovery of Mtb from sputum sediment samples was
shown to be more efficient if the samples are first diluted with either
EtOH alone or NaAc and EtOH together. Cultured Mtb were added to aliquots
of a pool of mixed respiratory sediments to obtain several different
titers, expressed in colony forming units (CFU) per PCR assay, and
NaAc/EtOH treatment was performed according to Method 1 described above.
Two hundred .mu.L aliquots were treated as indicated in Table 2. For EtOH
only, NaAc was omitted from the wash. For spin only, the wash was omitted
entirely, that is, the respiratory sediment alone, without EtOH or NaAc,
was centrifuged as for the first two treatments. The pelleted samples were
further processed and assayed for Mtb by PCR as outlined above. A no-spin
control, for which lysis reagent was added directly to the uncentrifuged
respiratory sediment, was included to show maximum recovery. Although many
respiratory sediment specimens interfere with polymer capture or PCR when
used without an ethanol wash, this particular sample was chosen for the
no-spin control because it did not interfere. Values given in Table 2 are
percentage of replicates that were PCR positive for Mtb; n=4 (*n=3;
**n=1).
The results shown in Table 2 indicate that cultured bacteria pellet more
efficiently from respiratory sediments in a medium containing EtOH or
NaAc/EtOH than with direct centrifugation in the sputum sediment without
EtOH (spin only). The respiratory sediment pool used was contaminated with
a very small number of Mtb organisms, not detected by culture, accounting
for the occasional PCR positive result in the zero CFU set.
TABLE 2
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Recovery of Cultured Mtb from Pooled Sputum Sediment
Treatment
Mtb NaAc + EtOH Spin No
(CFU) EtOH only only spin
______________________________________
0 0% 25% 0% 25%
2 75% 100% 0% 100%
5 100% 100% 50% 100%
10 100% 100% 50% 100%
50 100% 100% 75% 100%
100 100% 100% 100% 100%**
______________________________________
Example 3
Recovery of Mtb from Patient Samples
This experiment was identical to that in Example 2 except that the source
of Mtb was actual patient samples rather than culture. A pool of
respiratory sediment samples that had tested positive for Mtb by culture
was diluted with a pool of respiratory sediment samples that tested
negative for Mtb by culture. Two hundred microliter aliquots were treated
as in Example 2. Values given are percentage of replicates PCR positive
for Mtb; n=4. As in Example 2, the culture-negative pool contained levels
of Mtb that were detected by PCR. Therefore, any differences between
washes with and without NaAc were not observed. Nonetheless, a loss of
target was detected for samples pelleted without ethanol or sodium acetate
(spin only).
TABLE 3
______________________________________
Recovery of Mtb from Patient Samples
Dilution Treatment
of Mtb + NaAc + EtOH Spin No
pool EtOH only only spin
______________________________________
"neg" pool 100% 100% 50% 100%
10.sup.-7 100% 100% 25% 100%
10.sup.-6 100% 100% 75% 100%
10.sup.-5 100% 100% 25% 100%
10.sup.-4 100% 100% 100% 100%
10.sup.-3 100% 100% 100% 100%
______________________________________
Example 4
Comparison of Pure and Denatured Ethanol
It may be advantageous to use denatured ethanol instead of pure ethanol
because of cost and regulatory issues. Therefore, efficacy of recovery of
cultured mycobacteria from a respiratory sediment pool with pure ethanol
(200 proof) and denatured ethanol (90% ethanol, 5% methanol, 5%
isopropanol) was compared. Test samples were prepared and treated with
NaAc and EtOH as in Example 2, but denatured ethanol was substituted for
pure ethanol in one set of samples. Values given are percentage of
replicates PCR positive for Mtb; n=6. As in Examples 2 and 3, the
culture-negative pool is slightly contaminated with Mtb, however, the
results are identical for pure and denatured ethanol.
TABLE 4
______________________________________
Comparison of Pure Ethanol and Denatured Ethanol
Mtb Pure Denatured
(CFU) Ethanol Ethanol
______________________________________
0 17% 17%
0.5 83% 83%
1 100% 100%
2 100% 100%
5 100% 100%
10 100% 100%
______________________________________
Example 5
Comparison of EtOH alone with EtOH/NaAc
This experiment demonstrates that NaAc together with EtOH provide for more
efficient recovery and detection of mycobacteria from a subset of sputum
sediment samples. Test samples were prepared and treated with NaAc/EtOH or
EtOH alone, as in Example 2, except that a different, PCR-negative pool of
respiratory sediment was used, the volume of respiratory sediment was
increased to 300 .mu.L, and 700 .mu.L of the combined EtOH/NaAc reagent
(described in method 2 above) was used. Values given in Table 5 are
percentage of the replicates that were PCR positive for Mtb; n=4. As
shown, ethanol and sodium acetate together were more effective than
ethanol alone. Subsequent experiments demonstrated that DNA was recovered
efficiently by polymer capture when NaAc was included with the ethanol
treatment. Efficient polymer capture required a pH very close to 6.8. The
pH of some respiratory samples was 7.4 during polymer capture if NaAc was
omitted from the ethanol wash, and DNA was not captured efficiently.
However, the pH with a second aliquot of these same samples was 6.8 to 7.1
if NaAc was included with the EtOH, and DNA was captured efficiently. It
is evident that addition of NaAc adjusts the pH, and thereby, improves
recovery of Mtb DNA from this subset of clinical samples.
TABLE 5
______________________________________
Comparison of EtOH alone with EtOH/NaAc
Mtb EtOH EtOH/
(CFU) alone NaAc
______________________________________
0 0% 0%
0.25 0% 25%
0.5 25% 75%
1 50% 100%
2 0% 100%
5 25% 100%
______________________________________
Example 6
Detection of free Mtb DNA in Sputum Sediment Samples After Freeze-Thaw
This experiment demonstrates the presence of free Mtb DNA in previously
frozen sputum sediments. Three 200 .mu.L aliquots of culture-positive
sputum sediment samples, prepared as in Example 3, were centrifuged
without addition of EtOH/NaAc. The resulting supernatants were passed
through 0.2 .mu.m filters to remove residual bacteria. Both the filtered
supernatants, which would contain any free Mtb DNA, and the pellets, which
would contain intact Mtb, were processed further and assayed for Mtb DNA
by PCR as described above. Table 6 shows the percentage of replicates
(n=4) that were PCR positive for Mtb DNA. The data indicate that there is
significant free Mtb DNA in this sputum sediment sample, as much as ten
fold greater than the amount recovered from intact Mtb organisms. As shown
here and in Example 1, free DNA can be recovered with the method of the
present invention.
TABLE 6
______________________________________
Mtb DNA Detected in Respiratory Sediment Pellet and
Filtrate Without Wash
Dilution Pellet Filtrate
______________________________________
10.sup.-7 0% 0%
10.sup.-6 0% 0%
10.sup.-5 0% 100%
10.sup.-4 100% 100%
10.sup.-3 100% 100%
10.sup.-2 100% 100%
10.sup.-1 100% 100%
______________________________________
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one skilled in
the art from a reading of this disclosure that various changes in form and
detail can be made without departing from the true scope of the invention.
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